1. Field of the Invention
The invention relates to a device which makes it possible to compare the imaging characteristics and signal sensitivity of fluorescence detection systems and enables test-specific referencing of fluorescence signals. The invention also relates to a method for producing said device.
2. Description of the Related Technology
Frequently, biomedical tests are based on the detection of an interaction between a molecule or an affinity matrix whose identity or whose constitution is known (probe) and an unknown molecule to be detected, or unknown molecules to be detected (target molecule or target).
In modern tests, the probes, if they are molecules, are frequently immobilised on carriers in the form of a substance library in known quantity and position. Such devices are also called probe arrays or chips. Typically, a probe array comprises several so-called array elements which are the regions of a probe array in which a particular molecular probe is frequently immobilised in multiple copy. The sum of all occupied array elements thus constitutes the probe array.
Immobilisation of molecular probes in the form of a substance library on probe arrays makes it possible to analyse in parallel on several probes concurrently a sample which contains the target molecules to be detected. This makes possible a systematic analysis with a high throughput in a short time (high throughput screening, D. J. Lockhart, E. A. Winzeler, Genomics, Gene Expression and DNA Arrays, Nature 2000, 405, 827-836). For the purpose of producing the probe arrays, the probes are usually immobilised in a specified way on a suitable matrix, as described for example in WO 00/12575 (see e.g. U.S. Pat. No. 5,412,087, WO 98/36827) or synthetically produced (see e.g. U.S. Pat. No. 5,143,854).
In principle, furnishing proof of an interaction between the probe and the target molecule occurs as follows:
The probe or probes are attached in the specified way to a particular matrix in the form of a probe array. Subsequently, in a solution, the targets are brought into contact with the probes and incubated at defined conditions. If, due to complementary characteristics, the probe and the target molecule have an affinity to each other, then a specific interaction between the probe and the target takes place during incubation. The bond which occurs during this process is clearly more stable than the bond of target molecules on probes which are not specific to the target molecule. In order to remove non-specifically bonded target molecules, the system is washed with respective solutions, or heated, or is subjected to measures which have a correspondingly restrictive effect.
Furnishing proof of the specific interaction between a target and its probe can then be carried out by way of a multitude of methods which as a rule depend on the type of marker, such marker, depending on the design of the experiment, having been introduced into the target molecules or into the probe molecules during or after the interaction between the target molecule and the probe array. Such markers can for example include fluorescent groups, radioactive markers, enzymes or chemiluminescent molecules, with the method of proof to be used being governed by the type of marker (A. Marshall, J. Hodgson, DNA Chips: An Array of Possibilities, Nature Biotechnology 1998, 16, 27-31; G. Ramsay, DNA Chips: State of the Art, Nature Biotechnology 1998, 16, 40-44).
Depending on the substance library immobilised on the probe array, and depending on the chemical nature of the target molecules, by means of this test principle, interactions between nucleic acids, between proteins, and between nucleic acids and proteins can be investigated (for an overview, see F. Lottspeich, H. Zorbas, 1998, “Bioanalytik”, Spektrum Akademischer Verlag, Heidelberg/Berlin).
The following can be considered as possible substance libraries which can be immobilised on probe arrays or chips: antibody libraries, receptor libraries, peptide libraries and nucleic acid libraries. Nucleic acid libraries assume far and away the most important role, with DNA molecule libraries or RNA molecule libraries being used particularly frequently. In this context, probe-array-based analysis of interactions between nucleic acids follows the principles of the nucleic acid hybridisation technique (A. A. Leitch, T. Schwarzacher, D. Jackson, I. J. Leitch, 1994, “In vitro-Hybridisierung”, Spektrum Akademischer Verlag, Heidelberg/Berlin/Oxford).
Usually, proof of specific interactions between a probe and a target is furnished by fluorescence-optical evaluations because they are characterised by good sensitivity, versatility concerning the markers that can be used, and by the possibility of location-resolved and time-resolved detection of the interaction with comparatively little expenditure (above all in comparison with mass-spectroscopy methods) as well as by the elimination of irradiation exposure, the latter occurring when radioactive marking reagents are used. In addition, depending on the fluorophores used for marking, the excitation wavelength range and the detection wavelength range can be set.
However, in practical application, qualitative and quantitative fluorescence-optical evaluations are negatively affected by a number of factors connected to fluorescence spectroscopy per se, to the type of the fluorescence markers selected, and to the type and construction of the detection systems used. Such factors include above all non-specific background signals (signal noise) which arise as a result of intrinsic optical characteristics of the fluorescence markers (e.g. bleaching, quenching or fluorescence quenching of the dyes used); as a result of the physical-chemical characteristics of the probe or of the targets and their solutions (e.g. autofluorescence); as a result of fluctuations in the optical system (e.g. irradiation intensity of the light source, and extraneous light); and as a result of the construction and type of the detection systems used (e.g. autofluorescence of the assembly elements, capability of the detectors for spatial and temporal resolution, scattering, reflections).
Any assessment as to whether a measured fluorescence intensity represents a signal or merely forms part of signal noise thus requires elimination or minimisation of disturbing influences, as well as the use of devices and methods which make referencing of the measured fluorescence signals possible. Such devices are also referred to as a fluorescence calibration standard.
Efforts to minimise device-related signal noise result in very considerable technical expenditure on the construction of highly sensitive detectors which make possible both qualitative and quantitative evaluation of fluorescence signals. In particular, for evaluation during high throughput screening of probe arrays, which evaluation necessitates a certain degree of automation, specially adapted detection systems are required.
Fluorescence-optical read-outs of molecular probe arrays by means of standard epifluorescence structures, e.g. CCD (Charge Coupled Device) based detectors are used which for the purpose of qualitative differentiation of optical effects (scattering, reflection) achieve excitation of the fluorophores in the dark field (by way of reflected-light microscopy or transmitted-light microscopy) (C. E. Hooper et al., Quantitive Photone Imaging in the Life Sciences Using Intensified CCD Cameras, Journal of Bioluminescence and Chemiluminescence 1990, 337-344). In this process, imaging of the probe arrays takes place either by exposure or by rastering with the use of high-resolution optics. Complicated illumination optics and filter systems are necessary for minimising occurring autofluorescence, or for providing system-inherent optical effects such as illumination homogeneity, across the entire probe array.
Confocal scanning systems (described in U.S. Pat. No. 5,304,810) make it possible to evaluate fluorescence signals from selected planes of a sample. They are based on selecting the fluorescence signals along the optical axis by means of pinholes, which results in very considerable adjustment effort in relation to the samples, as well as necessitating the establishment of a powerful autofocus system. The technical implementation of such systems is highly complex, and the required components which include lasers, pinholes, (cooled) detectors (e.g. PMT, avalanche diodes, CCD systems), high-precision mechanical translation elements and optics, have to be integrated and optimised in relation to each other at considerable expense (described in U.S. Pat. Nos. 5,459,325, 5,192,980, 5,834,758).
Thus, detection systems are known with which the molecular interaction of a target comprising a fluorescence marker and a specific probe, as occurs e.g. in probe-array-based experiments, can be detected. Despite the very considerable technical expenditure described, which expenditure arises depending on the type and construction of the detection system used, for minimising signal noise, it is not possible to eliminate signal noise entirely. Therefore, qualitative and quantitative evaluation of measured fluorescence signals continues to necessitate referencing or calibration of the experiments and the detection devices by means of fluorescence calibration standards. Calibration of detection systems by means of fluorescence calibration standards is carried out, inter alia, in order to be able to make statements regarding the sensitivity of the spatial and temporal resolution and the geometric image aberrations, such as e.g. the curvature of field of the respective system.
Calibration of detection devices regarding their temporal resolution is necessary because, for the purpose of differentiating between the actual fluorescence signal (which is often long-lived) and autofluorescence signals (which are often short-lived), measuring of the signals has to be carried out across an extensive time period.
When using CCD detectors, it is necessary, by means of standards, e.g. to determine the linearity and sensitivity of the detector in the fluorescence wavelength range used, the spatial and temporal resolution, as well as the curvature of field (flat field determination) of the detector. Confocal detection systems require calibration with regard to the regions which are excited, or which contribute to the overall intensity.
Calibration of experiments by means of fluorescence calibration standards is necessary because the fluorophores which are used as markers are subjected to considerable fluctuations with regard to their fluorescence yields, due to the environmental conditions to which they are exposed (e.g. autofluorescence of solvent components, pH value, temperature, irradiation time), and thus absolute quantification e.g. of hybridisation yields on probe arrays is only possible with some reservations.
Calibration of various fluorescence detection systems by means of fluorescence calibration standards is also very important because only such calibration permits a comparison of fluorescence signals obtained in experiments, which fluorescence signals were obtained using different detection systems and different devices within a system (comparison above system level or above device level).
In the state of the art, various teachings are known which are intended to make it possible to calibrate fluorescence detection systems or fluorescence signals.
For the purpose of calibrating fluorescence detection devices, it is e.g. possible to use chips which comprise a fluorescent plastic layer. Such calibration standards are associated with the disadvantage that they do not allow calibration of the detection systems with regard to their spatial resolution or with regard to their dynamic characteristics across a wide fluorescence range. Similarly, determining the curvature of field e.g. of CCD detectors is not possible with these standards, because, due to the thickness of the chip, homogenisation of the fluorescence signal through the chip occurs. Thus, calibration of the influence which the geometric relationships have on the detection of fluorescence signals (influence which in particular in the case of different detection systems and principles can assume a decisive role), cannot be undertaken, either above device level or above system level.
When using CCD-based fluorescence detectors and in particular when carrying out excitation by funnel-shaped or beam-shaped lasers or multispectral illumination systems such as e.g. cold-light sources, alignment of the illumination homogeneity with the use e.g. of movable diffusing disks requires expensive calculation and image manipulation to define the flat field. From the state of the art, no fluorescence calibration standards are known which make it possible to define the flatfield for correcting the illumination homogeneity in such direct-imaging systems.
Furthermore, there are fluorescence calibration standards which are based on doped glass. These standards, too, are associated with a disadvantage in that they do not permit calibration of the detection systems with regard to their spatial resolution or with regard to their geometric characteristics, because, due to the dimensions of such standards, homogenisation of the signals takes place as a result of the volume of glass. Confocal systems in which only certain regions and layers along the optical axis are excited, or contribute to the overall intensity, and in which the problem of transmission losses through the structural layers which are lying above exists, also cannot be calibrated with regard to their geometric characteristics if such standards are used.
WO 01/06227 describes the manufacture of a fluorescence calibration standard based on microparticles or nanoparticles, and further describes their application, both for calibrating fluorescence detection systems and for referencing fluorescence intensity signals in fluorometric assays. These standards are also not suitable for calibrating the fluorescence detection systems with regard to their spatial resolution. Thus, any comparison above the device level in the case of signal intensity data obtained from experiments based on probe arrays is only possible with some reservations.
In order to be able to differentiate the short-lived background fluorescence (e.g. of autofluorescent solvent molecules) from the actual fluorescence signal, and in order to be able to reference the actual signal, long-term emitting marker substances can be used as a standard, with the signals of said marker substances being proven by time-resolved detection methods. Frequently, these marker substances are phosphorescent chelates of rare-earth metals (in particular those of europium or terpium). However, these substances are associated with a disadvantage in that they can only be excited with UV light sources. Furthermore, the chelates used are often unstable in aqueous form.
In order to achieve quantitative evaluation of molecular interactions in probe-array-based experiments by means of fluorescence measurements, and in order to achieve standardisation of such signals, for the purpose of calibrating or referencing, the experiments are carried out by dual-colouring of the probe molecules, e.g. by competitive hybridisation (M. Shena, D. Shalon, R. W. Davis, P. O. Brown, Quantitative monitoring of gene expression patterns with a complementary DNA microarray, Science, 1995, 220,467-70, and D. Shalon, S. J. Smith, P. O. Brown, A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe for hybridization, Genome Res., 1996, 6, 639-45). Since in such calibration standards the signals obtained by way of the reference molecules always depend on the concrete experimental conditions, it is difficult to carry out a quantitative comparison above the device level or above the system level.
The disadvantages of the fluorescence calibration standards known from the state of the art clearly show that there is a considerable requirement for devices which make it possible to calibrate fluorescence detection systems with regard to their spatial and temporal resolution, as well as with regard to their geometric and dynamic characteristics.
In addition, there is a considerable requirement for devices for referencing fluorescence signals which make possible comparisons above system level and above device level and/or comparison above the level of individual tests, of fluorescence signals e.g. of experiments based on probe arrays.